THE JOURNAL OF CELL BIOLOGY triggers aCa http://www.jcb.org/cgi/doi/10.1083/jcb.200604011 The JournalofCell Biology, Vol. 174,No.2,July17,2006221–229 © TheRockefellerUniversity Press SI, self-incompatibility. latrunculin B;PCD,programmedcell death;ROS,reactiveoxygenspecies; medium; Jasp, jasplakinolide;LatB, G-actin, globularactin;GM,germination Abbreviations usedinthispaper:CD,cytochalasin D;F-actin,fi Correspondence toVernonica E.Franklin-Tong: [email protected] S.G. ThomasandS.Huangcontributed equallytothispaper. increases inthecytosolic-free calciumconcentration([Ca pistil Sproteins(Foote etal.,1994)actasligands,triggering (Franklin-Tong andFranklin,2003).In of pollen S fertilization infl Self-incompatibility (SI)isoneofthesystemsthatprevent self- Introduction S 2 1 Steven G.Thomas, programmed celldeathinself-incompatiblepollen Actin depolymerizationissuffi Ca in incompatiblepollen(Franklin-Tong etal.,1993,1997). The stimuli (Swidzinskietal.,2002;Danon2004;Greenberg (Kuriyama andFukuda,2002)inresponsestoexternal death (PCD).Many examples ofPCDinplant development tions forthealterationtoactindynamics. quired toachieve this. This suggestedpossibleadditionalfunc- tube growth, itappearstobeagrossexcess over theamountre- depolymerization duringSIisclearlysuffi et al.,2000;Snowman etal.,2002). Although theextent ofF-actin of thepollenfi signals, reorganization andmassive, sustaineddepolymerization of incompatiblepollentubegrowth. Within afew minutesofSI (Jasp) andlatrunculinB,wedemonstratethatchangesin actin-stabilizing anddepolymerizingdrugsjasplakinolide dynamics wereimplicatedinregulatingPCD.Usingthe grammed celldeath(PCD).We investigatedwhetheractin tion oftipgrowth,actindepolymerization,andpro- Department ofBiologicalSciencesand Department B152TT, Edgbaston,Birmingham School ofBiosciences,UniversityBirmingham, England,UK locus; 2 + -mediated signalingnetwork resultsintherapidinhibition Unwanted cellsareusuallyremoved byprogrammed cell pollen. Inincompatible specifi elf-incompatibility (SI)preventsinbreedingthrough S- S specifi andpistil c recognitionandrejectionofincompatible lamentous actin(F-actin)isinduced(Geitmann 2 owering plants.SIiscontrolledbyamultiallelic + c pollenrejectionresultsfromtheinteraction signalingcascade,resultingintheinhibi- 1 S ShanjinHuang, determinantsthathave matchingalleles $8.00 3 Bindley BioscienceCenter, PurdueUniversity, West Lafayette,IN47907 Papaver rhoeas 2 ShutianLi, cient toinhibitpollen Papaver rhoeas lamentous actin; lamentous pollen,SI 1 ChristopherJ.Staiger, , the 2 + ] i ) signaling cascade(Janmey, 1998;Korichneva andHammerling, of actinfi Gourlay and Ayscough, 2005).Itispostulated thatthealteration 1999; Morley etal.,2003; Gourlayetal.,2004;forreview see Janmey, 1998;Korichneva andHammerling,1999;Raoetal., animal cells,dependingonthecelltype(Levee etal.,1996; cytoskeleton isadequatetoinducePCDinyeastandsome gests thateitherstabilizationordepolymerizationoftheactin depolymerization insignalingtoPCD.Recentevidence sug- Hall, 1998;Staiger, 2000),weexplored apossibleroleforactin signaling cascadesinbothanimalandplantcells(Schmidt the specifi Franklin-Tong, 2004). This provides aprecise mechanismfor pollen, whichinvolves acaspase-3–like activity (Thomasand ported thatSItriggersaPCDcascadeinincompatible in plantcells(Lametal.,2001;Schaller, 2004). We recentlyre- Despite this,thereisgoodevidence forcaspase-like activities logues have beenfoundinplants(Woltering etal.,2002). retically involve acaspase-3–like activity, nocaspasehomo- the fragmentationofnuclearDNA. Although PCDshouldtheo- strates, includingendogenousnucleaseinhibitors,resultingin by acaspasecascade. Activated caspasescleave numerous sub- and caspaseactivation. Inanimalcells,apoptosisismediated chrome documented. FeaturesofPCDinanimalcellsincludecyto- and Yao, 2004;van Doornand Woltering, 2005)have been mechanisms involvedinSI. cell andsignifi actin polymerizationstatusandinitiationofPCDinaplant resents thefi pollen wasalleviatedbypretreatmentwithJasp.Thisrep- like activity. Signifi initiating PCDin actin fi As theactincytoskeleton isamajortarget andeffector of cient toinduce lament levelsordynamicsplayafunctionalrolein c lament dynamicsinitiatesormodulatestheapoptotic leakagefromthemitochondria,DNA fragmentation, c destruction ofincompatiblepollen. 2,3 rst accountofaspecifi andVernonica E.Franklin-Tong cantly advancesourunderstandingofthe P. rhoeas cantly, SI-inducedPCDinincompatible pollen,triggeringacaspase-3– c causallinkbetween JCB: 1 P. rhoeas ARTICLE JCB

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on July 17, 2006 2006 17, July on www.jcb.org from Downloaded 1999; Rao et al., 1999; Morley et al., 2003; Gourlay et al., 2004), thereby committing cells to die (for review see Gourlay and Ayscough, 2005). Because PCD is triggered by SI in incom- patible P. rhoeas pollen (Thomas and Franklin-Tong, 2004), we hypothesized that early SI-induced actin depolymerization might play a role in acting as an upstream component in PCD activation. We have investigated this possibility using specifi c drugs to alter actin polymerization status in pollen tubes.

Results

Jasplakinolide stabilizes actin fi laments and alleviates SI-induced PCD Pollen tubes have a characteristic F-actin organization (Fig. 1 A), and SI induces the rapid depolymerization of actin fi laments and bundles (Fig. 1 B). To test the hypothesis that SI-stimulated PCD is triggered by actin depolymerization, we used jasplakino- lide (Jasp), which stabilizes actin fi laments and stimulates poly- merization (Bubb et al., 1994; Bubb et al., 2000) and has been shown to inhibit pollen tube growth (Cardenas et al., 2005). We found that 0.5 μM Jasp disrupted P. rhoeas pollen tube actin or- Downloaded from ganization, which induced actin fi lament bundling and aggregation (Fig. 1 C), and inhibited tip growth (unpublished data). The effect of Jasp on actin organization was essentially the opposite of that induced by SI. We examined whether transient treatments with Jasp might www.jcb.org “rescue” SI-induced pollen from PCD by counteracting the ac- Figure 1. Jasp alters actin organization and alleviates PCD induced by SI tin depolymerization. One of the hallmark features of PCD, in P. rhoeas pollen tubes. (A) Rhodamine-phalloidin staining shows normal DNA fragmentation, is triggered by SI and involves a caspase-3– organization of F-actin in an untreated P. rhoeas pollen tube. (B) Rhodamine- phalloidin staining shows major disruption of F-actin after 10 min of SI like activity (Thomas and Franklin-Tong, 2004). This was used induction. (C) Immunolocalization of actin alterations induced by 0.5 μM on July 17, 2006 as a marker for PCD and was assessed using TUNEL (Fig. 1, Jasp for 10 min shows dramatic rearrangement and stabilization of actin D and E). The induction of SI, which is caused by exposing fi laments. (D) DNA fragmentation, detected by TUNEL labeling, in a pollen tube treated with 1 μM LatB for 10 min. DAPI staining (right) shows that the incompatible pollen to S proteins for 30 min, produced high lev- TUNEL-positive signal (left) corresponds to nuclear DNA. (E) A typical con- els of DNA fragmentation of 55.2%, which was only slightly trol pollen tube shows no TUNEL-positive signal (left) corresponding to nu- lower than levels of 65.4% induced by SI for 8 h and signifi - clear DNA (right) despite overexposure revealing the background signal. < Bars, 10 μm. (F) Induction of SI for 30 min produced high levels of DNA cantly different from control levels of 16.8% (P 0.001; Fig. 1 F). fragmentation, which was only slightly lower than that achieved by an 8-h These data indicated that the interaction of incompatible pollen treatment (SI, 8 h). Consecutive (CON) treatment, in which 0.5 μM Jasp with S proteins for 30 min was suffi cient to trigger PCD. As we was added for 20 min to pollen tubes that had previously been treated for 10 min with S proteins, signifi cantly reduced the level of DNA fragmenta- had previously demonstrated that 10 min of SI stimulated a tion. Simultaneous (SIM) treatments of SI and Jasp for 30 min also signifi - 65% reduction in F-actin levels (Snowman et al., 2002), we al- cantly reduced levels of DNA fragmentation. Untreated pollen tubes lowed SI to progress for 10 min to ensure that a signifi cant level (control) produced low levels of DNA fragmentation, whereas 0.5 μM Jasp μ treatment for 30 min produced higher levels than controls but lower than of actin depolymerization had occurred and then added 0.5 M those induced by 30 min of SI. Values are mean percentages ± SEM (error Jasp for 20 min (see Pollen treatments). This consecutive treat- bars; n = 3). ment signifi cantly reduced DNA fragmentation (Fig. 1 F) by 32% (P = 0.04). Simultaneous treatment with SI and Jasp for 30 min also resulted in signifi cantly reduced levels of DNA To clarify whether actin depolymerization itself could induce fragmentation (33.8%; P < 0.001; Fig. 1 F). This demonstrates PCD, latrunculin B (LatB; Spector et al., 1983; Coue et al., that Jasp can alleviate SI-induced PCD. These data indicate that 1987) was used to depolymerize the actin cytoskeleton in grow- the actin depolymerization triggered by SI plays a functional ing P. rhoeas pollen tubes. LatB caused the rapid disruption role in the initiation phase of PCD in pollen tubes. of F-actin organization (Fig. 2, A and B) and the inhibition of pollen tube growth (unpublished data). To confi rm actin depoly- Latrunculin B reduces the level of F-actin merization, we measured F-actin levels (Gibbon et al., 1999; in P. rhoeas pollen Snowman et al., 2002) after 10 min of LatB treatments. LatB To further investigate whether actin polymerization status is in- caused the depolymerization of pollen tube actin fi laments in volved in the initiation of PCD in pollen, we attempted to mimic a concentration-dependent manner (Fig. 2 C). Treatment with the actin depolymerization induced by SI without the involve- 0.1, 1, and 10 μM LatB caused signifi cant reductions (P < 0.001) 2+ ment of other SI initiation signals, such as changes to [Ca ]i. in F-actin levels when compared with controls. Treatment with

222 JCB • VOLUME 174 • NUMBER 2 • 2006 Figure 3. Alterations in the actin polymerization status trigger a caspase- Downloaded from Figure 2. LatB and Jasp induce DNA fragmentation. (A) Rhodamine- like/DEVDase activity upstream of DNA fragmentation. (A) LatB- and Jasp- phalloidin staining of F-actin organization in a normally growing pollen induced DNA fragmentation is dependent on a caspase-like/DEVDase μ tube. (B) Rhodamine-phalloidin labeling of a pollen tube treated with 1 M activity. LatB and Jasp (white bars) induced high levels of DNA fragmenta- LatB for 10 min showing the reorganization and depolymerization of tion. Pretreatment with DEVD signifi cantly reduced the levels of DNA frag- μ F-actin. Bars, 10 m. (C) Treatment of growing pollen tubes with LatB for mentation (black bars), whereas pretreatment with YVAD (crosshatched 10 min caused the concentration-dependent depolymerization of F-actin. bars) had no signifi cant effect on DNA fragmentation induced by LatB and ± Data are mean F-actin levels SEM (error bars) as a percentage of the Jasp. (B) DEVD signifi cantly reduced levels of DNA fragmentation in pollen www.jcb.org = DMSO controls (n 3). (D) Treatment with LatB for 8 h triggered DNA tubes treated with LatB. (A and B) Data are mean percentages of TUNEL- fragmentation. DMSO controls showed only low levels of DNA fragmentation. positive nuclei ± SEM (error bars) from three independent experiments. (E) Treatment with Jasp for 8 h triggered DNA fragmentation. (D and E) Data (B and C) Untreated pollen, white bars; + 1 μM LatB for 10 min, black ± are mean percentages of TUNEL-positive nuclei SEM from four bars; + 1 μM LatB for 60 min, crosshatched bars. (C) DEVD had no effect

(D) or three (E) independent experiments. on LatB-induced actin depolymerization induced in growing pollen tubes; on July 17, 2006 similar levels of F-actin were measured in the presence and absence of DEVD. Data are means ± SEM from four independent experiments. 1 μM LatB, which induced 61.5% F-actin depolymeriza- tion, gave a similar reduction to that stimulated by SI (69%; Snowman et al., 2002). assumptions regarding the lack of Jasp and LatB side effects, the results indicate that changes in actin fi lament dynamics can Actin depolymerization is suffi cient induce DNA fragmentation. to induce PCD A hallmark feature of apoptosis and PCD is the involve- To test whether LatB-induced actin depolymerization could ment of caspases, which are responsible for initiating and exe- trigger PCD, pollen tubes were treated with LatB, and levels of cuting cell death. We showed previously that SI-induced PCD is DNA fragmentation were assessed after 8 h. LatB stimulated DNA mediated by a caspase-3–like/DEVDase activity (Thomas and fragmentation in a concentration-dependent manner (Fig. 2 D). Franklin-Tong, 2004). Therefore, we tested whether the DNA The incidence of DNA fragmentation at all concentrations fragmentation stimulated by LatB and Jasp involved such an of LatB was signifi cantly different from control pollen tubes activity. We used the tetrapeptide Ac-DEVD-CHO (DEVD), (P < 0.001) and increased from 6.9% in controls to 30.7% in which is a caspase-3 inhibitor (Garcia-Calvo et al., 1998; the presence of 0.01 μM LatB and to 75.8% with 10 μM LatB Richael et al., 2001; Danon et al., 2004; Thomas and Franklin- (Fig. 2 D). Assuming that LatB does not have side effects, this Tong, 2004). Pollen was pretreated with either DEVD or the indicated that actin depolymerization triggered DNA fragmen- caspase 1 inhibitor Ac-YVAD-CHO (YVAD), which acts as a tation independently of other SI signals. We also examined negative control, for 1 h before the addition of LatB or Jasp for 8 h. whether actin fi lament stabilization or net assembly of la-fi Pollen tubes pretreated with DEVD had signifi cant (65.1– ments from the profi lin–actin pool might also induce PCD in 71.6%) reductions in the levels of DNA fragmentation com- pollen. Jasp stimulated DNA fragmentation in a concentration- pared with controls without pretreatment (P < 0.001; Fig. 3 A). dependent manner (Fig. 2 E). The incidence of DNA fragmen- Pretreatment with YVAD had no signifi cant effect on the levels of tation in Jasp-treated pollen tubes increased from 10.4% in DNA fragmentation induced by LatB or Jasp (P = 0.2; Fig. 3 A). control samples to 58% in the presence of 1 μM Jasp (Fig. 2 E). Because DEVD can prevent LatB- or Jasp-induced DNA As Jasp binds the phalloidin-binding site of actin, we could not fragmentation, this implies that both actin depolymerization quantify actin polymer levels for these treatments. Given the and stabilization or polymerization can stimulate the activation

ACTIN DEPOLYMERIZATION INDUCES PCD IN POLLEN • THOMAS ET AL. 223 Table I. Quantifi cation of actin polymer level and DNA fragmentation

Treatment Actin polymer level at Actin polymer level Percent TUNEL-positive nuclei end of treatment after washing

DMSO 100 100 16.4 ± 1.4 0.1 μM LatB for 5 min ND ND 19.5 ± 2.2 0.1 μM LatB for 10 min 77.0 ± 8.9 125.9 ± 7.3 24.3 ± 0.6 0.1 μM LatB for 30 min ND ND 57.1 ± 8.2 0.1 μM LatB for 60 min 49.8 ± 5.5 107.3 ± 9.0 64.0 ± 4.0 1 μM LatB for 5 min ND ND 58.5 ± 9.9 1 μM LatB for 10 min 46.9 ± 3.8 93.5 ± 10.1 56.4 ± 11.8 1 μM LatB for 30 min ND ND 68.2 ± 6.2 1 μM LatB for 60 min 30.8 ± 4.2 70.1 ± 5.2 70.5 ± 6.3

Results are means ± SEM (n = 3). The actin polymer level is expressed as a percentage of the amount measured in the control (i.e., control is 100%). TUNEL data are expressed as the percentage of nuclei showing TUNEL labeling.

of a caspase-like enzyme upstream of DNA fragmentation. either 10 or 60 min gave a high incidence of DNA fragmenta- These data indicate that changes in actin fi lament dynamics are tion even though F-actin levels returned to normal after washing. suffi cient to induce a caspase-like (DEVDase) activity that re- This indicates that during a brief 10-min period of actin depoly- sults in PCD in pollen. merization to this level, an irreversible “decision-making” step is made, pushing pollen into PCD. Because a DEVDase/ caspase- Transient F-actin depolymerization like activity is involved, this implicates actin depolymeri zation in Downloaded from is suffi cient to induce PCD PCD initiation. Treatments that did not reduce F-actin to this To examine whether transient changes in actin fi lament levels level (e.g., 0.1 μM LatB for 10 min) had a low incidence of or dynamics could serve as a signal involved in the initiation of DNA fragmentation (24.3%), which was only slightly higher PCD or whether sustained alterations are required, “washout” than the control level (P = 0.02). Therefore, we can say with experiments were conducted using LatB treatments, as this some confi dence that here, the reduced incidence of DNA frag- most closely mimicked the effect of SI. Pollen tubes were mentation was the result of a transient actin depolymerization www.jcb.org treated with 0.1 and 1 μM LatB for 10 or 60 min, after which that was insuffi cient to cause PCD. Thus, the threshold for PCD the drug was washed out and the incidence of DNA fragmenta- induction is somewhere between a 23 and 46.9% reduction in tion was assessed. Actin fi lament levels were determined at the F-actin levels for as little as 10 min. Our data indicate that on July 17, 2006 end of the LatB incubation period to demonstrate that depoly- <50% actin depolymerization for 10 min is suffi cient to induce merization had taken place and also after washouts to establish PCD, achieving levels of DNA fragmentation very similar to whether repolymerization had occurred. As expected, F-actin that induced by 8 h of LatB (P = 0.4; NS). Notably, this thresh- levels were reduced in a concentration- and time-dependent old level of actin depolymerization required to initiate PCD manner (Table I). The level of actin polymer after treatment using LatB is somewhat lower than the 58 and 69% reduction in with 0.1 μM LatB was reduced by 23.0% (P = 0.03) at 10 min F-actin levels resulting from 5- and 10-min SI inductions, and was further reduced by 50.2% (P < 0.001) by 60 min. The respectively (Snowman et al., 2002). higher concentration of 1 μM LatB reduced actin levels by 53.1% (P < 0.001) at 10 min and by 69.2% (P < 0.001) at LatB-induced PCD is mediated 60 min. After washouts, with the exception of the 1-μM 60-min by a caspase-3–like activity treatment, F-actin levels returned to similar levels as found in As LatB treatments appeared to mimic the SI–PCD response, untreated pollen, demonstrating that F-actin was only depoly- we examined whether LatB-induced DNA fragmentation relied merized transiently (Table I). Pollen tubes treated with 0.1 μM on a caspase-like/DEVDase activity similar to that induced by LatB also resumed normal growth (unpublished data). SI (Thomas and Franklin-Tong, 2004). DNA fragmentation was DNA fragmentation correlated with the duration and extent normally induced by 1-μM LatB treatments, whereas DEVD of actin depolymerization. After 5- and 10-min treatments with pretreatment prevented this (Fig. 3 B), and levels of DNA 0.1 μM LatB, the effects were similar, and only small increases fragmentation were not signifi cantly different from untreated in DNA fragmentation were detected (P = 0.3; NS; Table I). samples (P = 0.13). These data provide strong evidence that even With longer treatments of 0.1 μM LatB, DNA fragmentation in- quite transient LatB-induced actin depolymerization is suffi - creased to 57.1% at 30 min (P = 0.02) and to 64.0% at 60 min cient to induce a caspase-3–like/DEVDase activity upstream of (P = 0.001). The 1-μM LatB treatments induced higher levels of DNA fragmentation. Because DEVD had no signifi cant effect DNA fragmentation than those found in the controls for all four on the amount of actin depolymerization caused by LatB (Fig. time points (P < 0.001). However, there was no signifi cant dif- 3 C), this established that DEVD does not interfere with the ference between the four treatments (P = 0.6; Table I), indicating action of LatB. that a threshold level of DNA fragmentation had been triggered. We have also used the caspase-3 substrate Ac-DEVD- Taking these F-actin quantifi cation and DNA fragmenta- AMC to establish this caspase-like activity more directly. Pollen for tubes treated with 1 μM LatB for 6 h exhibited an increase in %50ف tion data into account, reducing F-actin levels to

224 JCB • VOLUME 174 • NUMBER 2 • 2006 DEVDase activity of 54 ± 5% (n = 4) compared with con- trol samples containing germination medium (GM) only. This level of caspase activity was similar to that induced by either SI or 0.5-μM Jasp treatments (unpublished data). Thus, LatB- stimulated pollen extracts exhibited markedly increased levels of caspase-3–like activity, indicating cleavage of the substrate by a DEVDase activity. The activation of caspases commits the cell to die; therefore, our data implicate alterations in actin de- polymerization as playing a functional role in stimulating or regulating the PCD cascade in pollen.

Inhibition of tip growth does not induce Figure 4. Actin depolymerization induces PCD, which can be alleviated by altering the actin polymerization status. The effect of counteracting LatB- PCD in pollen tubes induced depolymerization with Jasp was assessed by the extent of DNA Because both LatB and Jasp inhibit tip growth (Gibbon et al., fragmentation. Untreated pollen tubes (control) produced low levels of 1999; Vidali et al., 2001; Snowman et al., 2002; Cardenas et al., DNA fragmentation; treatment with 0.1 μM LatB for 30 min produced in- μ 2005), we wished to establish that PCD induction was not indi- creased levels of DNA fragmentation, and 0.5 M Jasp for 30 min pro- duced increased but lower levels of DNA fragmentation. With combined rectly caused by the cessation of tip growth. We used caffeine, LatB-Jasp treatments (consecutive [CON] treatments [10 min with 0.1 μM which perturbs tip growth (Pierson et al., 1996) but does not LatB and then 0.5 μM Jasp for a further 20 min] and simultaneous [SIM] μ μ affect the actin cytoskeleton of pollen tubes (Geitmann et al., treatments [0.1 M LatB and 0.5 M Jasp for 30 min], the level of DNA fragmentation was signifi cantly reduced compared with that observed with 2000; Snowman et al., 2002). Pollen tubes treated with 3 mM LatB or Jasp alone. Data are mean percentages of TUNEL-positive nuclei ± caffeine for 10 or 60 min did not signifi cantly alter F-actin lev- SEM (error bars) from four independent experiments. Downloaded from els compared with controls (112 ± 12 and 112 ± 2%, respec- tively; n = 3; P = 0.06). Caffeine-treated pollen tubes had low DNA fragmentation levels that were not signifi cantly different Our study provides a signifi cant advance in our under- from controls (15 ± 3% at 10 min, P = 0.2; and 23 ± 4% at standing of the mechanisms involved in early SI and of the initi- 60 min, P = 0.06; n = 3). This demonstrates that the LatB/Jasp- ation of PCD in plant cells because very little is known about the induced DNA fragmentation is not merely a consequence of the early events involved in PCD in plants. Our data provide evi- www.jcb.org inhibition of growth but is caused by changes in actin polymer dence that relatively transient but substantial F-actin depolymer- levels or assembly dynamics. ization can trigger PCD, which is mediated by a caspase-3–like activity. We previously demonstrated that the SI response in on July 17, 2006 Jasp counteracts LatB-induced PCD P. rhoeas pollen results in a 69% reduction in F-actin levels within We also investigated whether counteracting the effect of LatB- 10 min (Snowman et al., 2002) and, independently, that SI also induced depolymerization with Jasp might prevent progression induces PCD (Thomas and Franklin-Tong, 2004). In this study, into PCD. Pollen tubes were treated in a similar manner to the we have demonstrated that a transient reduction in F-actin levels SI experiment. They were fi rst treated with 0.1 μM LatB for of <50%, which is independent of SI induction, is suffi cient to 10 min to ensure that actin depolymerization had occurred; induce PCD. This provides evidence for a link between the sig- 0.5 μM Jasp was then added for 20 min, after which the drugs naling cascades involving actin dynamics and PCD, showing were washed out. Thus, the total length of time that the pollen that actin depolymerization is suffi cient to induce a caspase- was exposed to LatB for this consecutive treatment was 30 min. like/DEVDase activity resulting in PCD. Furthermore, together This treatment resulted in a 45% reduction (P = 0.002) in the with previous data (Snowman et al., 2002), they demonstrate level of DNA fragmentation compared with LatB alone for 30 that during an incompatible SI response, the degree of actin de- min (Fig. 4). We also performed treatments whereby LatB and polymerization is more than adequate to act as an intermediary Jasp were added simultaneously; this had a similar signifi cant signal to PCD. Because Jasp can alleviate SI-induced DNA effect with a 51.7% (P = 0.007) reduction in the incidence of fragmentation, presumably by interfering with or counteracting PCD to 19.8%, which was not signifi cantly different (P = 0.2) the actin depolymerization induced by SI, this provides further, from control levels of 11.3% (Fig. 4). These data show that Jasp more direct evidence for the involvement of actin depolymeriza- can counteract the actin depolymerization induced by LatB and, tion in SI-induced PCD. Thus, rapid depolymerization of the thereby, rescues pollen from entry into PCD. actin cytoskeleton induced by SI not only inhibits pollen tip growth but also acts upstream of a signaling cascade that is in- Discussion volved in initiating PCD. Our data support the hypothesis that the F-actin depolymerization and PCD observed during SI func- The actin cytoskeleton has been identifi ed as a major target and ef- tion together in a signaling network that prevents incompatible fector of signaling cascades in both animal and plant cells (Schmidt pollen from affecting fertilization. and Hall, 1998; Staiger, 2000). SI may be viewed as the triggering Although LatB or cytochalasin D (CD) has previously of a signaling cascade that takes the cell into PCD. In this study, been reported to implicate actin depolymerization in PCD dur- we provide the fi rst account of a causal link between actin polymer ing embryogenesis in plants (Smertenko et al., 2003), the drug levels or dynamics and the initiation of PCD in a plant cell. treatments extended over a long period of time (6 d), and

ACTIN DEPOLYMERIZATION INDUCES PCD IN POLLEN • THOMAS ET AL. 225 disruption of the actin cytoskeleton for this length of time will F-actin, prevented cisplatin-mediated actin depolymerization almost certainly result in defects in embryogenesis; this in itself and apoptosis in porcine kidney proximal tubule cells (Kruidering could result in PCD. Thus, although the data suggested a possi- et al., 1998). This is exactly what we have demonstrated here; ble involvement of the actin cytoskeleton in PCD, they provided Jasp effectively reverses the effects of S-protein treatment, no clear evidence for a functional role for actin depolymerization presumably by stabilizing actin fi laments against depolymeriza- in PCD. tion or counteracting the calcium-induced disassembly. Our fi ndings establish a fundamentally important role for Therefore, it would appear that a precise level of actin the actin cytoskeleton as a sensor of cellular stress that is com- polymer or exact fl ux of subunits through the polymer pool is mon to many eukaryotic cells, as various links between changes necessary for normal growth and cell viability of pollen (Gibbon in actin dynamics and PCD induction have previously been re- et al., 1999; Vidali et al., 2001); when this is substantially per- ported for animals and yeast (Levee et al., 1996; Janmey, 1998; turbed, PCD is initiated. It has been proposed that it is the al- Korichneva and Hammerling, 1999; Suria et al., 1999; Morley teration of actin dynamics (i.e., the rate of actin polymeriza tion et al., 2003; Gourlay et al., 2004). Several studies have shown and depolymerization) that modulates the transduction of the that alterations to the actin cytoskeleton (either stabilization or apoptotic signal (Morley et al., 2003). The alterations to actin depolymerization, depending on the cell type involved) can play providing the sensory mechanism could involve either changes a functional role as an effector in the initiation of PCD. For in polymer levels, changes in the fl ux of actin through the fi la- example, CD resulted in DNA fragmentation and caspase-3 ment pool, or both. However, although in some mammalian activity in Jurkat T cells and hippocampal neurons (Suria et al., cells, alterations to F-actin status alone (either polymerization 1999), and Jasp enhanced and accelerated apoptosis induced by or depolymerization, depending on the cell type) are suffi cient cytokine withdrawal in the IL-2–dependent T cell line CTLL-20 to induce apoptosis, in other cell types, other apoptotic stim- and induced a caspase-3 activity (Posey and Bierer, 1999). uli are required in addition to drug treatments affecting actin Downloaded from Because these data demonstrate that presumed changes to actin dynamics (Korichneva and Hammerling, 1999; Posey and polymer levels or dynamics can stimulate caspase-3 activity, Bierer, 1999; Morley et al., 2003). Thus, there are likely to be which is an effector caspase triggered early in PCD, they pro- quite important and subtle differences in the mechanisms oper- vide evidence that actin depolymerization or stabilization is suf- ating to initiate apoptosis/PCD in different cell types. One no- fi cient to act as an intermediary signal early in the PCD signaling table and important difference between plant pollen and yeast cascade. Other studies in yeast and some mammalian cells have or mammalian cells is the high ratio of globular actin (G-actin) www.jcb.org also shown that decreasing actin fi lament turnover (by using to F-actin in actively growing cells. Measurements from maize Jasp to stabilize actin) induces apoptosis (Posey and Bierer, and P. rhoeas pollen indicate that just 5–10% of the total ac- 1999; Rao et al., 1999; Odaka et al., 2000; Gourlay et al., 2004). tin protein is present in the fi lamentous form (Gibbon et al., on July 17, 2006 Thus, it has been proposed that altering actin dynamics or the 1999; Snowman et al., 2002). In comparison, budding yeast rates of assembly and disassembly rather than the level of poly- cells are considered to have the majority of the total actin pool meric actin in cells is suffi cient to signal the initiation of apoptosis in fi lamentous form (Karpova et al., 1995). These observations (Janmey, 1998; Korichneva and Hammerling, 1999; Rao et al., further suggest that certain differences in apoptosis initiation 1999; Suria et al., 1999; Odaka et al., 2000; Cioca and Kitano, and/or responses to cytoskeletal drugs may relate to the endo- 2002; Kim et al., 2003; Morley et al., 2003; Gourlay et al., 2004; genous balance between monomer and polymer in different for review see Gourlay and Ayscough, 2005). eukaryotic cells. Interestingly, although actin stabilization stimulates PCD Importantly, our study, which used washouts to achieve in yeast, actin depolymerization has the reverse effect, leading short-term actin depolymerization or stabilization, is, to our to increased viability (Gourlay et al., 2004), which is not the knowledge, the fi rst to demonstrate that transient (10–60 min) case in pollen. This suggests that although there are clear parallels, alterations to actin dynamics can trigger PCD. Most of the the specifi c mechanisms are likely to differ between plants, aforementioned studies have used rather long-term treatments animals, and yeast. This is borne out by several studies with with Jasp or LatB for between 8 and 48 h to induce apoptosis mammalian cells, in which actin depolymerization can induce (Posey and Bierer, 1999; Suria et al., 1999; Gourlay et al., or promote apoptosis (Korichneva and Hammerling, 1999; 2004). To our knowledge, this is the only study to quantify the Posey and Bierer, 1999; Suria et al., 1999; Cioca and Kitano, changes in F-actin levels required to trigger PCD. Only two 2002; Morley et al., 2003), whereas actin stabilization can in- other studies attempted to quantify the actin polymerization hibit or delay apoptosis (Korichneva and Hammerling, 1999; status (Rao et al., 1999; Morley et al., 2003). One of these studies Genesca et al., 2006). Indeed, it has been shown in Jurkat T cells reports that measurements after CD treatment of CTLL-20 and that either increased actin depolymerization or polymerization Jurkat T cells indicated an increase in the amount of monomeric using CD or Jasp can enhance apoptosis (Posey and Bierer, or G-actin and a decrease in the amount of F-actin, suggesting 1999; Morley et al., 2003). This appears to be the situation in the promotion of actin fi lament disruption and depolymeriza- pollen, in which either LatB or Jasp can induce PCD. Further- tion (Morley et al., 2003). However, no data are shown. The more, it has recently been shown that Jasp can alleviate apopto- other study (Rao et al., 1999) measured G- and F-actin levels sis in ischemic kidney cells; ischemia normally correlates with after camptothecin-induced apoptosis in HL-60 cells and found a substantial depolymerization of the actin cytoskeleton (Genesca that G-actin was decreased in TUNEL-negative cells at 2 h, in- et al., 2006). Similarly, phalloidin treatment, which stabilizes dicating that polymerization was increased early in apoptosis.

226 JCB • VOLUME 174 • NUMBER 2 • 2006 In TUNEL-positive cells (in which PCD had already occurred), (Snowman et al., 2002) at 25°C. Pollen was grown for 1 h before any G-actin levels were increased, indicating depolymerization. treatments were applied. For SI treatments, recombinant proteins were produced by cloning These data are consistent with camptothecin- and Jasp-induced the nucleotide sequences specifying the mature peptide of the S1, S3, and actin polymerization occurring before PCD. Our data go further S8 alleles of the S gene (pPRS100, pPRS300, and pPRS800) into the ex- than this because we have estimated the levels of actin depoly- pression vector pMS119 as described previously (Foote et al., 1994). Expression and purifi cation of the proteins were performed as described merization required to induce PCD in pollen. In this study, we previously (Kakeda et al., 1998). SI was induced by adding recombinant show that 50% depolymerization of the actin cytoskeleton for S proteins (fi nal concentration of 5 μg/ml) to pollen growing in vitro as de- just 10 min is suffi cient to trigger substantial numbers of cells scribed previously (Snowman et al., 2002). For DNA fragmentation experiments with actin inhibitors, LatB or to undergo PCD. Jasp (both from Calbiochem) were added to growing pollen tubes at vari- The alterations to actin could involve either changes in ous concentrations. Pollen was grown for a total of 8 h, after which it was polymer levels, changes in the fl ux of actin through the fi lament fi xed in 2% PFA and assayed for DNA fragmentation (see next section). For washout experiments, after short treatments with LatB or Jasp, pollen tubes pool, or both. Because virtually no biochemical analysis of ac- were washed extensively in GM (six washes of 5 ml for 5 min each). Pollen tin dynamics leading to PCD has been performed, it would be of tubes were resuspended in GM and incubated for the remainder of the 8-h considerable interest to establish which mechanisms are in- period. Controls were comprised of the addition of DMSO at a fi nal con- centration of 0.1% (vol/vol). volved and whether they vary depending on cell type. Similarly, Caffeine (Sigma-Aldrich) treatments consisted of the addition of the several different actin-binding proteins have been suggested to drug to pollen tubes at a fi nal concentration of 3 mM; pollen was incu- play a role during the initiation or execution phases of PCD, but bated for 10 or 60 min before washouts as described above. Controls were comprised of the addition of GM alone. cause and effect relationships are diffi cult to decipher. We re- For the SI-Jasp experiments, pollen tubes were subjected to two an- cently identifi ed pollen gelsolin, PrABP80, which is a calcium- tagonistic treatments with incompatible S proteins and Jasp. To ascertain a stimulated actin fi lament severing and depolymerizing protein suitable Jasp treatment, washout experiments were conducted. Treatments of 0.5 μM Jasp for 10 and 30 min followed by washouts only stimulated that could potentially be involved in SI-mediated actin depoly- ± ± levels of DNA fragmentation between 31.2 2.5 and 33.0 0.4%, Downloaded from merization (Huang et al., 2004). This is of interest, as gelsolin is respectively. This provided a suitable concentration of Jasp for attempts to implicated in modulating apoptosis in animal cells (for review counteract the PCD induced by LatB. Consecutive SI-Jasp treatments con- sisted of the addition of S proteins for 10 min followed by the addition of see Kwiatkowski, 1999). 0.5 μM Jasp for 20 min. Simultaneous treatments involved the addition of How exactly actin polymerization status can affect apopto- S proteins and Jasp at the same time and incubation for 30 min. Immedi- sis induction is not yet established in any organism. However, ately after each treatment, the pollen tubes were washed extensively with GM as described above and were incubated for the remainder of the 8-h www.jcb.org there is evidence that changes to actin dynamics interact with period before assaying for DNA fragmentation. Control treatments were apoptotic signaling cascades. It has been suggested that disrup- comprised of 30 min in 0.1% DMSO or 0.5 μM Jasp. The LatB-Jasp experi- tion of the cytoskeleton might promote the release of caspases, ments were performed in a similar manner. enabling their activation or, alternatively, disrupting mitochon- DNA fragmentation assay on July 17, 2006 dria and causing the release of cytochrome c, caspases, or cas- Fixed pollen tubes were labeled with a Deadend Fluorometric TUNEL kit pase activators (Suria et al., 1999). In mammalian cells, there is (Promega) according to the manufacturer’s instructions. Pollen tubes were scored for DNA fragmentation (50 tubes per treatment; n = 3) using a evidence that either actin depolymerization (Suria et al., 1999) or fl uorescence microscope (T300; Nikon) and a 60× plan-Apo 1.4 NA oil stabilization (Posey and Bierer, 1999; Cioca and Kitano, 2002; objective (Nikon). Capture and analysis of images was performed at 20°C Genesca et al., 2006) can induce caspase-3 activation; actin de- using a camera (SenSys KAF1400-G2; Photometrics) and an image analy- sis system (Quips PathVysion; Applied Imaging). Composite images were polymerization using CD has been shown to regulate nitric prepared using Adobe Photoshop 8.0. oxide–stimulated apoptosis by modulating PI3-kinase, PKC, and MAPK signaling (Kim et al., 2003). In yeast, it has been pro- Caspase assays posed that the actin cytoskeleton could act as a regulator for re- To test whether DNA fragmentation was dependent on a caspase-like activity, pollen was grown in the presence of 100 μM DEVD or YVAD (Calbiochem) active oxygen species (ROS) release from mitochondria, as Jasp for 1 h before the addition of the cytoskeletal drugs for 8 h. Samples were can stimulate changes in the levels of ROS (Gourlay et al., 2004). then tested for DNA fragmentation using TUNEL. Inhibition of DNA We have shown here that actin depolymerization or polymeriza- fragmentation by DEVD was taken as evidence for caspase-3–like activity. We also used the fl uorogenic caspase-3 substrate Ac-DEVD-AMC tion is suffi cient to induce a caspase-3–like activity in pollen. (Calbiochem) to establish this caspase-like activity more directly. Pollen Whether ROS is involved in SI is not yet known, but it is known tubes were treated with 1 μM LatB and 0.5 μM Jasp or GM for 6 h, and that the rapid disruption of mitochondria is triggered by SI protein extracts were made from them by grinding in extraction buffer (50 mM sodium acetate and 10 mM L-cysteine, pH 6.0). 10 μg of total protein (Thomas and Franklin-Tong, 2004). Furthermore, we have previ- was incubated with 50 μM Ac-DEVD-AMC at 27°C, and fl uorescence was ously shown that a MAPK, p56, is activated by SI (Rudd et al., monitored at 460 nm using a time-resolved fl uorescence plate reader 2003). These provide possible targets for signaling links between (FLUOstar OPTIMA; BMG LABTECH). Fluorescence at 480 nm indicated cleavage of the substrate by a DEVDase activity. Relative fl uorescence units the actin cytoskeleton and PCD in pollen. Thus, our data clearly were expressed as the percent increase relative to the control after 4 h. demonstrate that actin acts as a sensor for signals and that its dy- namics play a key role in signaling to initiate PCD in plant cells. F-actin imaging Pollen was grown on GM medium solidifi ed with 1.2% wt/vol agarose, treated with actin inhibitors at various concentrations and different times as Materials and methods described in the fi gure legends and text, and fi xed with 400 μM 3-maleimi- dobenzoic acid N-hydroxysuccinimide ester (MBS; Pierce Chemical Co.) for Pollen treatments 6 min followed by 2% PFA for 40 min. Pollen tubes were washed three times Pollen of P. rhoeas, the fi eld poppy, was germinated and grown in vitro in in actin-stabilizing buffer (100 mM Pipes, pH 6.8, 1 mM MgCl2, 1 mM liquid GM (0.01% H3BO3, 0.01% KNO3, 0.01% Mg(NO3)2-6H2O, CaCl2, and 75 mM KCl) and permeabilized with TBS-Tween-DTT (50 mM Tris, 0.036% CaCl2-2H2O, and 13.5% sucrose) as described previously pH 7.4, 200 mM NaCl, 0.05% Tween 20, and 5 mM DTT) for 15 min.

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Gametophytic self-incompatibility 0.5-μm sections, and each section was the average of three Kalman scans. inhibits pollen tube growth using different mechanisms. Trends Plant Sci. Images were exported, and composite images were prepared using Adobe 8:598–605. Photoshop 8.0. Franklin-Tong, V.E., J.P. Ride, N.D. Read, A.J. Trewavas, and F.C.H. Franklin. For Jasp-treated pollen, pollen tubes were grown, treated with 0.5 μm 1993. The self-incompatibility response in Papaver rhoeas is mediated by Jasp for 10 min, fi xed with PFA and MBS, and prepared for immuno- cytosolic-free calcium. Plant J. 4:163–177. 2+ localization. Samples were washed three times in actin-stabilizing buffer Franklin-Tong, V.E., G. Hackett, and P.K. Hepler. 1997. Ratio-imaging of [Ca ]i and once in MES buffer (5 mM MES, pH 5.0, 5 mM EGTA, and 0.4 M in the self-incompatibility response in pollen tubes of Papaver rhoeas. mannitol). Cell walls were digested with 0.1% cellulase and 0.1% macero- Plant J. 12:1375–1386. zyme in MES buffer containing 0.1 mM PMSF for 10 min. Pollen tubes Garcia-Calvo, M., E.P. Peterson, B. Leiting, R. Ruel, D.W. Nicholson, and N.A. were washed once in MES, washed twice in TBS, permeabilized in 0.1% Thornberry. 1998. Inhibition of human caspases by peptide-based and Triton X-100 in TBS for 10 min, and washed in TBS + 1% BSA. Samples macromolecular inhibitors. J. Biol. Chem. 273:32608–32613. were then incubated with a 1:250 dilution of anti-actin antibody (Gibbon Geitmann, A., B.N. Snowman, A.M.C. Emons, and V.E. Franklin-Tong. et al., 1999) overnight at 4°C. Unbound primary antibody was washed 2000. Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas. Plant Cell. out, and the pollen tubes were incubated for 2 h at RT in anti–rabbit FITC 12:1239–1252. antibody (1:200 dilution). 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